• 제목/요약/키워드: microplate

검색결과 151건 처리시간 0.025초

Optimization of Tri-enzyme Extraction Procedures for the Microbiological Assay of Folate in Red Kidney Bean and Roasted Peanut Using Response Surface Methodology

  • Choi, Young-Min;Eitenmiller, Ronald R.;Kim, Seon-Hee;Lee, Jun-Soo
    • Food Science and Biotechnology
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    • 제18권1호
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    • pp.31-35
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    • 2009
  • Total folate content was determined by microbiological assay using Lactobacillus casei spp. rhamnosis (ATCC 7469) with a 96-well microplate technique. Using roasted peanut and red kidney beans as representative legume samples, response surface methodology (RSM) was supplied to optimize the trienzyme procedures for the determination of folate in legumes. After response surface regression (RSREG), the second-order polynomial equation was fitted to the experimental data. Ridge analysis showed that the optimal digestion times were <2 hr for $Pronase^{(R)}$ and $\alpha$-amylase, and <5 hr for conjugase to obtain maximal folate values for legume samples. This study confirms that established digestion times for cereal products (AOAC Method 2004.05) of 3 for protease and 2 hr for $\alpha$-amylase are applicable to legumes. Conjugase treatment can be reduced to 5 from 16 hr and the conjugase level to 5 from 20 mg per sample, providing significant cost saving.

Optimization of Cu, Hg and Cd removal by Enterobacter cloacae by ferric ammonium citrate precipitation

  • Singh, Rashmi R.;Tipre, Devayani R.;Dave, Shailesh R.
    • Advances in environmental research
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    • 제3권4호
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    • pp.283-292
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    • 2014
  • Iron precipitating organisms play a significant role in the formation of ferric hydroxide precipitate, which acts as strong adsorbent for toxic metal. In this respect four different iron precipitating cultures were isolated from Hutti gold mine surface winze water sample on citrate agar medium. The best isolate was screened out for metal removal study on the basis of fast visual iron precipitation. The selected isolate was identified as Enterobacter sp. based on routine biochemical tests and Biolog GN microplate results and as Enterobacter cloacae subsp. dissolvens by 16S rRNA gene sequence analysis (GenBank accession number EU429448). Influence of medium composition, medium initial pH, the influence of inoculum size, effect of various media and ferric ammonium citrate concentration were studied on metal removal in shake flask experiments. Under the optimized conditions studied, E. cloacae showed $94{\pm}2$, $95{\pm}2$ and $70{\pm}2%$ of cadmium, copper and mercury removal from a simulated waste in shake flask studies. In lab scale column reactor more than 85% of copper and mercury removal was achieved.

Elastase Inhibitor 생성 방선균의 탐색 및 동정 (Screening and Identification of Actinomycetes Producing Novel Elastase Inhibitor)

  • 이병규;강희일;이계준
    • 미생물학회지
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    • 제33권4호
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    • pp.225-231
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    • 1997
  • Elastase 저해물질(EI) 생성균주로 토양분리주 SMF-11을 선별하였다. 분리주 SMF-11는 매끈한 표면의 rectiflexible 및 spiral의 포자 사슬을 동시에 형성하고 세포벽 성분으로 LL-DAP, ios- 및 anteiso-fatty acid($C_{14}-C_{17}$), 세포벽 아미노산으로 alanine, glutamic acid, glycine을 가지고 있어 방선균속으로 분류되었다. 종 수준의 동정을 위하여 화학적 시험과 50가지의 단위형질에 대한 특성을 조사하고 TAXON program으로 수리분류한 결과, SMF-11 분리주는 방선균 주군집 61의 중심균주인 Streptomyces lavendulae로 동정하였다.

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Sandwich ELISA 방법을 이용한 생물공학의약품에 잔류하는 숙주유래단백질의 검출법개발 (Determination of Remained Host Derived Proteins in the Commercially Available Biotechnological Products Using a Sandwich Enzyme Linked Immunosorbent Assay Method)

  • 성혜윤;최규실;김창민;민홍기;용군호
    • Biomolecules & Therapeutics
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    • 제2권2호
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    • pp.120-125
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    • 1994
  • We obtained the total protein antibodies of Saccharomyces cerevisiae KCTC 1720 and Escherichia coli K-12 from the rabbit and the guinea pig to determine the host-derived proteins which may be remained in biotechnological products. The protein concentration of rabbit antibodies was 4.05 mg/mι in the case of yeast, 7.14 mg/mι in the case of E. coli and that of guinea pig antibodies was 1.90 mg/mι in the case of yeast, 7.17 mg/mι in the case of E. coli, respectively. To determine remained host-derived proteins in biotechnological products which produced by the hosts, S. cerevisiae or E. coli, we used a sandwich enzyme linked immunosorbent assay method in 96 well microplate. When the method applied to determine the remained host-derived proteins in commercial biotechnological products, it detected less than 3.5 ng/vial in human growth hormone, less than 1 ng/vial in hepatitis B vaccine and interferon-${\gamma}$ and 2~23 ng/vial in interferon-$\alpha$. The method can be used to determine the remained host-derived protein in biotechnological products.

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신생아 대사질환 선별검사에서 발견된 갈락토스혈증의 감별진단 (Differential Diagnosis of Galactosemia Detected by Neonatal Screening)

  • 최성윤;송웅주;임한혁;길홍량;김숙자
    • 대한유전성대사질환학회지
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    • 제13권2호
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    • pp.89-97
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    • 2013
  • Purpose: We retrospectively investigated individuals who hadbeen identified by neonatal screening as potential galactosemia patients to determine the etiology of galactosemia. Methods: One hundred fifty-three patients referred to Korea Genetics Research Center due to high galactose level detected by neonatal screening test between February 2005 and May 2013 were examined. Galactose and galactose-1-phosphate levels were measured by using a fluoro metric microplate reader. Lactose free diet was initiated immediately after confirmed by urine Clinitest. If reducing sugar was negative, we employed abdominal sonogram and echocardiogram to check for possible porto-systemic shunt. Results: Fifteen patients were diagnosed with galactosemia. One patient had galactokinase (GALK) deficiency; four had UDP galactose-4-epimerase (GALE) deficiency; two had citrin deficiency; and four had porto-systemic shunt. Two had unknown causes of galactosemia. Conclusion: In addition to genetic defects of GALT, GALK and GALE, citrin deficiency or porto-systemic shunt could also cause galactosemia. It is crucial to carry out differential diagnosis to determine the cause of galactosemia.

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Synthesis, Docking Study and In-vitro Evaluation of Anti-Tuberculosis Activity of Tri Substituted Imidazoles Containing Quinoline Moiety

  • Sahana, S.;Vijayakumar, G.R.;Sivakumar, R.;Sriram, D.;Saiprasad, D.V.
    • 대한화학회지
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    • 제66권3호
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    • pp.194-201
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    • 2022
  • A simple, efficient, and cost-effective method has been employed for the synthesis of 2,4,5-trisubstituted imidazole derivatives (3a-j) containing quinoline substituent at 2nd position. Title compounds were obtained by multicomponent reaction (MCR), involving aryl substituted 1,2-diketone, quinoline carbaldehyde and ammonium acetate in the presence of acetic acid solvent under mild reaction conditions. The newly synthesized quinoline containing imidazole derivatives were confirmed through FT-IR, 1H-NMR, 13C-NMR and mass spectral analysis. In-vitro microplate alamar blue assay (MABA) to determine the MIC (minimum inhibitory concentration) values against Mycobacterium tuberculosis H37Rv was performed for the synthesized compounds. The synthesized compounds exhibited activity against Mycobacterium tuberculosis and among which compounds, 3d, 3f and 3i showed good activity. The highest activity was showed with compound 3i. The anti-mycobacterial activity results are well correlated with the computational molecular docking analysis, which was performed for the synthesized compounds prior to the evaluation of the activity.

쑥부쟁이 추출물의 아세틸콜린에스테라제 저해 및 항산화 활성 (Acetylcholinesterase Inhibitory and Antioxidant Properties of Aster yomena Extract)

  • 배종섭;김태훈
    • 대한본초학회지
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    • 제24권4호
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    • pp.121-126
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    • 2009
  • Objectives : To evaluate the radical scavenging and acetylcholinesterase (AChE) inhibitory activities of the ethylacetate (EtOAc)-soluble portion of a methanolic extract of Aster yomena, three different assay systems were performed. Methods : The antioxidant activity of A. yomena extract was tested as its capacity to scavenging free radicals of DPPH and $ABTS^+$, which has been widely used to evaluate the antioxidant activity of natural products from plant sources. AChE inhibitory activity was tested against mouse brain AChE by spectrophotometric method of Ellman using ELISA microplate reader. Results : The methanolic extract of A. yomena was fractionated and the EtOAc-soluble portion showed significant AChE inhibitory and free radical scavenging effects. Also the EtOAc-soluble portion revealed the highest phenolic contents as compared to the other extracts. Conclusions : These results indicate that phenolic compounds may be important constituents that give rise to the anti-AChE and antioxidative activities of A. yomena extract. Further phytochemical studies on this plant, for nutraceutical or pharmaceutical application, are warranted.

A Highly Sensitive Indirect Enzyme-Linked Immunosorbent Assay (ELISA) Based on a Monoclonal Antibody Specific to Thermal Stable-Soluble Protein in Pork Fat for the Rapid Detection of Pork Fat Adulterated in Heat-Processed Beef Meatballs

  • Sol-A Kim;Jeong-Eun Lee;Dong-Hyun Kim;Song-min Lee;Hee-Kyeong Yang;Won-Bo Shim
    • 한국축산식품학회지
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    • 제43권6호
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    • pp.989-1001
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    • 2023
  • Processed foods containing pork fat tissue to improve flavor and gain economic benefit may cause severe issues for Muslims, Jews, and vegetarians. This study aimed to develop an indirect enzyme-linked immunosorbent assay (iELISA) based on a monoclonal antibody specific to thermal stable-soluble protein in pork fat tissue and apply it to detect pork fat tissue in heat-processed (autoclave, steam, roast, and fry) beef meatballs. To develop a sensitive iELISA, the optimal sample pre-cooking time, coating conditions, primary and secondary dilution time, and various buffer systems were tested. The change in the iELISA sensitivity with different 96-well microtiter microplates was confirmed. The detection limit of iELISA performed with an appropriate microplate was 0.015% (w/w) pork fat in raw and heat-treated beef. No cross-reactions to other meats or fats were shown. These results mean that the iELISA can be used as an analytical method to detect trace amounts of pork fat mixed in beef.

경남지역에서 발생한 가금티푸스의 역학적 특성 및 진단방법에 대한 비교 시험 (Epidemiological characteristics on fowl typhoid outbreak in Kyongnam province and comparison of diagnostic methods for identification of salmonella gallinarum)

  • 최유정;김도경;김용환
    • 한국동물위생학회지
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    • 제23권4호
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    • pp.349-360
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    • 2000
  • An epidemiological survey was conducted to investigate fowl typhoid outbreaks in Kyungnam province of Korea. The causative agent, salmonella gallinarum was isolated from 68 chicken samples of tentatively diagnosed fowl typhoid cases occurred during the period from January 1996 to September 1999. Comparative studies were also carried out to evaluate the diagnostic methods for detection of S gallinam The results obtained were as follows; 1. Of the 68 cases of tentatively diagnosed fowl typhoid, 56 (82%) cases were determined as fowl typhoid by biochemical test and pathological findings. The other 12 (18%) cases were determined as paratyphoid. 2. Fowl typhoid outbreaks occur continuously all seasons in the year, however the incidence was remarkably increased from May to September. 3. The frequency of incidence of fowl typhoid in terms of regional distribution was relatively high in egg-laying hens facilities, and the mode of transmission is likely to be either egg-to-egg or lateral transfer by wild birds or rats. 4. All of 18 isolates from 56 cases were identified as S gallinarum by biochemical and serological test. 5. Antimicrobial drug susceptibility test against 18 isolates showed that the isolates were highly susceptible to ASH, CZ, CF and GM (above 90%), whereas those strains were 100% resistant to EM, NA and PC. 6. S gallinarum rfbS gene was targeted to be amplified by PCR for comparative detection of S gallinarum in the experimentally infected chickens. The amplified 720bp DNA fragment, which is specific in D serogroup strains of S enterica subspecies was confirmed by agarose gel electrophoresis. 7. A comparison made between fecal culture and PCR-method revealed that later-method was relatively higher in detection rate than that of former method for S gallinarum. 8. Comparison of currently applied methods, rapid serum agglutination test (RST) and microplate agglutination test (MAT), with experimentally infected chickens were made to evaluate sensitivity of detection by neutralizing antibody titration. Both methods detected neutralizing antibodies from the challenged chickens of 5 day post infection. However, positive reactions were determined after 7 and 9 days post infection by MAT and RST, respectively.

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Enterocarpam-III Induces Human Liver and Breast Cancer Cell Apoptosis via Mitochondrial and Caspase-9 Activation

  • Banjerdpongchai, Ratana;Wudtiwai, Benjawan;Pompimon, Wilart
    • Asian Pacific Journal of Cancer Prevention
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    • 제16권5호
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    • pp.1833-1837
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    • 2015
  • An aristolactam-type alkaloid, isolated from Orophea enterocarpa, is enterocarpam-III (10-amino-2,3,4,6-tetramethoxyphenanthrene-1-carboxylic acid lactam). It is cytotoxic to various human and murine cancer cell lines; however, the molecular mechanisms remain unclear. The aims of this study were to investigate cytotoxic effects on and mechanism (s) of human cancer cell death in human hepatocellular carcinoma HepG2 and human invasive breast cancer MDA-MB-231 cells compared to normal murine fibroblast NIH3T3 cells. Cell viability was determined by MTT assay to determine $IC_{10}$, $IC_{20}$ and $IC_{50}$ levels, reactive oxygen species (ROS) production with 2',7'-dichlorohydrofluorescein diacetate and the caspase-3, -8 and -9 activities using specific chromogenic (p-nitroaniline) tetrapeptide substrates, viz., DEVD-NA, IETD-NA and LEHD-NA and employing a microplate reader. Mitochondrial transmembrane potential (MTP) was measured by staining with 3, 3'-dihexyloxacarbocyanine iodide ($DiOC_6$) and using flow cytometry. The compound was cytotoxic to HepG2 and MDA-MB-231 cells with the $IC_{50}$ levels of $26.0{\pm}4.45$ and $51.3{\pm}2.05{\mu}M$, respectively. For murine normal fibroblast NIH3T3 cells, the $IC_{50}$ concentration was $81.3{\pm}10.1{\mu}M$. ROS production was reduced in a dose-response manner in HepG2 cells. The caspase-9 and -3 activities increased in a concentration-dependent manner, whereas caspase-8 activity did not alter, indicating the intrinsic pathway activation. Enterocarpam-III decreased the mitochondrial transmembrane potential (MTP) dose-dependently in HepG2 cells, suggesting that the compound induced HepG2 cell apoptosis via the mitochondrial pathway. In conclusion, enterocarpam-III inhibited HepG2 and MDA-MB-231 cell proliferation and induced human HepG2 cells to undergo apoptosis via the intrinsic (mitochondrial) pathway and induction of caspase-9 activity.